A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.
A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.
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一种亲和标记胰腺胆囊收缩素受体的新探针，对其结构进行了微小修改。
DOI：
10.1111/j.1432-1033.1989.tb15128.x
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
N. Vaysse
中科院分区：
文献类型：
--
作者：
D. Fourmy;P. López;S. Poirot;J. Jiménez;M. Dufresne;L. Moroder;S. P. Powers;N. Vaysse
对肽受体的生物化学研究最常在亲和标记（肽-受体）复合物上进行。必然地，假设共价（肽-受体）复合物表现为天然受体。该假设的有效性取决于亲和标记技术和用于生化表征的分析方法的分辨率。我们设计了一种新的亲和标记探针，以尽量减少亲和标记的胆囊收缩素（CCK）受体蛋白内发生的结构修饰。探针为~（125）I-ASD-[Thr 28，Ahx 31]CCK-25-33（~（125）I-ASD-[Thr 28，Ahx 31]CCK-25-33）。它是通过将光活化化学物质通过其N-末端偶联至[Thr 28，Ahx 31]CCK-25-33而获得的。所得肽经HPLC纯化，并在氯胺T存在下放射性碘化。~（125）I-ASD-[Thr 28，Ahx 31]CCK-25-33的结合具有时间和温度依赖性，可逆性。在25 ℃下，60 min后达到稳态水平，38 min后达到半最大解离。[Thr 28，Ahx 31]CCK-25-33和L-364-718拮抗剂抑制结合，IC 50分别为0.4 nM和0.9 nM。用~（125）I-ASD-[Thr 28，Ahx 31]CCK-25-33对胰腺质膜进行光亲和标记，鉴定出一种Mr为85，000 - 100，000的糖蛋白，它保留在固定化的麦胚凝集素上。通过内切蛋白酶Glu-C的酶裂解产生Mr 30，000 - 34，000的主要片段。用125 I-DTyr-Gly-[Ahx 28，31，pNO 2 Phe 33]CCK-26-33（Ahx，2-氨基己酸; pNO 2 Phe，p-硝基苯丙氨酸）光亲合标记相同的糖蛋白，CCK-26- 33是一种内在探针，其光不稳定基团位于胆囊收缩素的结合结构域中。~（125）I-ASD-[Thr 28，Ahx 31]CCK-25-33是研究胆囊收缩素受体的一个潜在的生物学和生化学工具。
Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.
分析胆囊收缩素受体短探针标记的胰腺质膜糖蛋白亲和力的碳水化合物组成。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Pearson,RK;Miller,LJ;Hadac,EM;Powers,SP
通讯作者：
Powers,SP
胰腺中胆囊收缩素受体的异质性。
DOI：
10.1016/0006-291x(87)91419-7
发表时间：
1987
影响因子：
3.1
作者：
Madison,LD;Jamieson,JD;Rosenzweig,SA
通讯作者：
Rosenzweig,SA
胆囊收缩素 (CCK)-胃泌素家族受体的固有光亲和标记探针。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Powers,SP;Fourmy,D;Gaisano,H;Miller,LJ
通讯作者：
Miller,LJ